1. Field of the Invention
This invention relates to the conversion of certain sub-types of blood type A erythrocytes into type O cells to render for use in tranfusion therapy. More especially, this invention relates to a process for the conversion of certain sub-type A erythrocytes into type O cells under conditions whereby the cells do not lose their cellular functions, are suitable for the adsorption and release of oxygen whereby the cells can be transfused in the manner of type O blood. This invention also relates to the products obtained by the conversion of such erythrocyte sub-types into type O cells.
2. Discussion of Prior Art p As is well known in the transfusion therapy, it is necessary to match the blood type of the recipient with the type of blood available in the blood bank. Thus, for instance, a recipient of type A blood can only be safely transfused with type A blood. The exception to this is type O blood, the erythrocytes of which can be safely transfused into type A, type B and type A, B recipients as well as O recipients.
In the operation of a blood bank or other facility which accumulates whole blood or at least the red cell component thereof, it is necessary to maintain supplies of each type of blood. It has not heretofore been possible to maintain only O type blood because there is a paucity of O type donors. O type donor blood has therefore been used largely for O type recipients. On the other hand, a majority of donors have A, B or AB blood and there can exist from time to time, an excess of these types of blood. It has become desirable, therefore, to adjust the supply to the demand. Specifically, it has been desired to convert A, B or AB type blood to an O type blood type--a universal donor.
The ABO blood group system was the first to be discovered and is the one of greatest importance from the point of view of blood transfusion. Individuals of blood types A, B and O express A, B and H antigens respectively. These antigens are not only found in the red cells, but on the surfaces of all endothelial and most epithelial cells as well. In addition, glycoproteins having A, B and H antigenicity are also found in the tissue fluids and secretions of those individuals who have the ability, inherited as a Mendelian dominant character, to secrete these blood group substances, or factors as they are termed.
While the blood group substances are glycoproteins, the A B H active material obtained from the cell membranes appear to be glycolipids and glycoproteins.
Considerable work has been done to determine the structures of the A B H determinants. It was found that the blood group specificity of the entire molecule, which may contain one or more carbohydrate chains, attached to a peptide backbone, is determined by the nature and linkage of those monosaccharides situated at the non-reducing ends of these chains. The most important sugar for each specificity, often referred to as the immuno-dominant or immuno-determinant sugar, was found to be as follows: for H antigen, fucose; for A antigen, N-acetyl-galactosamine; and for the type B antigen, galactose. More recently, studies with A B H active glycolipids obtained from erythrocyte cell membranes also show the presence of the same immunodominant sugars at the reducing ends of the carbohydrate chains, attached to adjacent sugars by the same linkages. The carbohydrate chains are, in turn, linked either to protein or the ceramide, which is embedded in the lipid bi-layer of the membrane. The length of the carbohydrate moiety may vary and it may have either a straight or branched structure. Thus far, four variants of blood group active A glycolipid, two of B and three of H, have been isolated from the erythrocyte cell membrane.
Through these studies, it was theorized that one could convert a type A or type B antigen into a type H antigen, corresponding to a type O cell by removal of one of the monosaccharide groups pendent from the cell.
It has recently been demonstrated that the galactose molecule responsible for B antigenicity can be enzymatically removed from type B cells, thus converting them to type O erythrocytes under conditions that maintain cell viability and membrane integrity. Furthermore, it has been shown that one ml quantities of such enzymatically coverted cells survive not only normally in the circulation when returned to the original type B donor, but also when such quantities are transfused to A and O recipients whose immune systems would not tolerate unconverted type B cells (Goldstein, J., Siviglia, G., Hurst, R., Lenny L. and Reich L., Science 215 168, 1982). See also U.S. Pat. No. 4,330,619 disclosing conversion of B erythrocytes to O erythrocytes using .alpha.-galactosidase.
Specifically, in the case of A antigen it was also postulated that the N-acetylgalactosamine moiety of the type A-antigen could be removed enzymatically whereby the type A antigen would be converted to type H antigen. A previous attempt has been made to accomplish such an enzymatic conversion and produce transfusable quality cells. See Levy and Animoff, Journal of Biological Chemistry Vol. 255, No. 24, Dec. 25, 1980, pages 11737-42. This was unsuccessful, however, because only a partial removal of A antigenicity was achieved by the bacterial enzyme employed since these treated cells were still agglutinated (up to sixteen fold dilution) with human anti A antiserum. Such cells are not viable for transfusion to type O and B recipients becasue they would produce the same effect as untreated type A erythrocytes, inducing transfusion reactions and being destroyed by the recipient's immune system. Furthermore, this bacterial enzyme is contaminated with significant amounts (0.1%) of another enzyme known as sialidase. Treatment of erythrocytes with sialidase results in their premature aging, i.e. the uncovering of cryptic antigens resulting in agglutination of these cells by all human sera which leads to their rapid removal from the circulation following transfusion. Thus, even if all A antigenicity had been removed, the sialidase present in the bacterial enzyme preparation renders such treated cells unfit for transfusion.
There are three recognized major subtypes of blood type A known as A.sub.1, A intermediate or A.sub.int and A.sub.2. There are both quantitative and qualitative differences distinguishing the subtypes. A.sub.1 cells have more antigenic A sites, i.e. terminal N-acetylgalactosamine residues, than A.sub.int cells which in turn have more than A.sub.2 erythrocytes. Qualitatively the transferase enzymes responsible for the formation of A antigens differ biochemically from each other in A.sub.1, A.sub.int and A.sub.2 individuals. This suggests a genetic basis for three subtypes, namely that the A genetic locus is subdivided into three common genetic sites, each representing a different subtype and each site coding for its own specific transferase enzyme.
The number of A.sub.int and A.sub.2 individuals vary widely in different populations. For example, the frequency of A.sub.2 is approximately 30% of A.sub.1 in Caucasians (British) and less than 1% in Orientals (Japanese). Whereas the frequency of A.sub.int is only about 1-2.5% of A in Caucasians, 0.5% in Orientals but ranges from 1-32% in blacks.
It is an object of this invention, therefore, to provide a process in which the terminal moiety of the A-antigenic determinant of stroma from certain sub-type, of A and AB type cells can be removed while leaving the red cells intact so that the resultant composition can be used in transfusion therapy. Specifically, it is an object of this invention to convert sub-types A and AB cells to O type cells whereby the cells remain intact and undergo little if any hemolysis and the resultant composition can be used in transfusion therapy. Specifically, it is an object of this invention to covert certain sub-types of A and AB cells to O type cells whereby the cells remain intact and undergo little if any hemolysis and the resultant composition can be used in transfusion therapy. The invention is directed to the conversion of A (intermediate) and A.sub.2 cells and the corresponding A B cells into O cells. These cells which are converted into O cells are hereinafter designated as "A.sub.int -A.sub.2 cells", it being understood that the term embraces corresponding B cells, e.g. A.sub.2 B cells.